Gloria Fernández-Lorente

IMMOBILIZATION OF LIPASES BY SELECTIVE ADSORPTION ON HYDROPHOBIC SUPPORTS

The preparation of immobilized derivatives of lipases that may be useful to develop industrial processes of organic synthesis is an exciting field of research in which three main features have to be simultaneously considered: (a) immobilized derivatives have to be compatible with very different reaction requirements (e.g. continuous adjustment of pH with concentrated alkali, use of aqueous media or organic solvents, etc.); (b) Sometimes, some activity/stability properties of lipases should be improved during immobilization; and (c) because of a complex mechanism of action, lipases are poorly active in the absence of hydrophobic interfaces. In this paper, we will review different approaches for lipase immobilization mainly related to the further use of immobilized derivatives to carry out enantio and regioselective hydrolysis in high water-activity systems. Special emphasis is paid to the selective adsorption of lipases on tailor-made strongly hydrophobic support surfaces. This new immobilization procedure is based on the assumption that the large hydrophobic area that surrounds the active site of lipases is the one mainly involved in their adsorption on strongly hydrophobic solid surfaces. Thus, lipases recognize these surfaces similarly to those of their natural substrates and they suffer interfacial activation during immobilization. This immobilization method permits: (a) promote a dramatic hyper-activation of most of lipases after their immobilization. That is, adsorbed lipases show very enhanced esterase activity in the absence of additional hydrophobic interfaces; (b) promote highly selective adsorption of lipases, at very low ionic strength, from impure protein extracts. That is, we can associate immobilization and purification of lipases; (c) promote interesting improvements of enantioselectivity after immobilization; and (d) promote a strong but reversible immobilization that enables us to recover these expensive supports after inactivation of immobilized lipases.

Epoxy Sepabeads: A Novel Epoxy Support for Stabilization of Industrial Enzymes via Very Intense Multipoint Covalent Attachment

Sepabeads‐EP (a new epoxy support) has been utilized to immobilize‐stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 μmol/mL). These features should permit a very intense enzyme‐support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three‐step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds‐fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 °C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to “facilitate” the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).

General Trend of Lipase to Self-Assemble Giving Bimolecular Aggregates Greatly Modifies the Enzyme Functionality

Three microbial lipases (those from Candida rugosa, Humicola lanuginosa, and Mucor miehei) have been found to exhibit a tendency to form bimolecular aggregates in solution even at very low enzyme concentrations (44 microg/mL) in the absence of a detergent, as detected by gel filtration. The monomolecular form of the enzymes was found as unique only at low enzyme concentration and in the presence of detergents. However, in the case of the lipase B from Candida antarctica, no bimolecular form could be identified even at enzyme concentrations as high as 1.2 mg/mL in the absence of detergent. It has been stated that bimolecular and monomolecular structures display very different functional properties: (i) the enzyme specific activity decreased when the lipase concentration increased; (ii) the bimolecular form was much more stable than the monomeric one yielding a higher optimal T (increasing between 5 and 10 degrees C) and higher stability in inactivation experiments (the dimer half-life became several orders of magnitude higher than that of the monomer); (iii) the enantioselectivity depended on the enzyme concentration even after immobilization. For example, with use of the lipase from H. lanuginosa, the enantiomeric excess of the remaining ester in the hydrolysis of fully soluble ethyl ester of (R,S)-2-hydroxy-4-phenylbutanoic acid varied from 4 to 57 when the concentrated or diluted enzyme immobilized on PEI support, respectively, was used. It seems that the bimolecular structure of lipases might be formed by two open lipase molecules (interfacially activating each other) in very close contact and hence with a very altered active center.

Activation of Bacterial Thermoalkalophilic Lipases is Spurred by Dramatic Structural Rearrangements.

The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60–75 °C and pH 8–10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the α6- and α7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the α6-helix and the incorporation of short side chain residues to the α6 C-terminal end. All these structural changes are stabilized by the Zn2+-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate.

Modulation of the enantioselectivity of Candida antarctica B lipase via conformational engineering. Kinetic resolution of (±)-α-hydroxy-phenylacetic acid derivatives

Abstract The modulation, via immobilization and engineering the reaction medium, of the enantioselectivity exhibited by the lipase from Candida antarctica B (CABL) in the hydrolysis of α-hydroxy-phenylacetic acid derivatives is shown. The enzyme was purified and immobilized using different protocols to obtain immobilized enzyme preparations with different orientations and micro-environments. The catalytic properties (activity, specificity, enantioselectivity) of the resulting derivatives were found to be quite different from each other. The enantioselectivity ( E value) strongly depends on the type of derivative and the conditions employed. Thus, the enzyme immobilized on cyanogen bromide (CNBr) presented E =7.4, while the PEI derivative yielded E =67 in the hydrolysis of α-hydroxy-phenylacetic acid methyl ester under similar conditions. Moreover, the enantioselectivity of the PEI derivative decreased from 67 to 14 on lowering the reaction temperature from 25 to 4°C at pH 5, while the E of some other derivatives improved significantly under similar experimental changes. Similar changes in the E values were observed in the hydrolysis of ( RS )-2-butyroyl-2-phenylacetic acid. Using this substrate, the interfacially adsorbed enzyme (octadecyl) afforded an E value of only 2 at pH 5, while the glutaraldehyde derivative presented a high enantioselectivity ( E >400) under all conditions studied. The corresponding ( S )-ester and ( R )-acid were obtained with excellent enantiomeric excess using the glutaraldehyde derivative, while using the interfacially immobilized one there was no appreciable enantioselectivity. Thus, using differently immobilized derivatives and different experimental conditions, lipase enantioselectivity could vary from negligible to up to 400. The experimental conditions were also found to have varying effects on the different lipase derivatives.

Affinity chromatography of polyhistidine tagged enzymes: New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.

Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose.

In this paper, the stabilization of a lipase from Bacillus thermocatenulatus (BTL2) by a new strategy is described. First, the lipase is selectively adsorbed on hydrophobic supports. Second, the carboxylic residues of the enzyme are modified with ethylenediamine, generating a new enzyme having 4-fold more amino groups than the native enzyme. The chemical amination did not present a significant effect on the enzyme activity and only reduced the enzyme half-life by a 3-4-fold factor in inactivations promoted by heat or organic solvents. Next, the aminated and purified enzyme is desorbed from the support using 0.2% Triton X-100. Then, the aminated enzyme was immobilized on glyoxyl-agarose by multipoint covalent attachment. The immobilized enzyme retained 65% of the starting activity. Because of the lower p K of the new amino groups in the enzyme surface, the immobilization could be performed at pH 9 (while the native enzyme was only immobilized at pH over 10). In fact, the immobilization rate was higher at this pH value for the aminated enzyme than that of the native enzyme at pH 10. The optimal stabilization protocol was the immobilization of aminated BTL2 at pH 9 and the further incubation for 24 h at 25 degrees C and pH 10. This preparation was 5-fold more stable than the optimal BTL2 immobilized on glyoxyl agarose and around 1200-fold more stable than the enzyme immobilized on CNBr and further aminated. The catalytic properties of BTL2 could be greatly modulated by the immobilization protocol. For example, from (R/S)-2- O-butyryl-2-phenylacetic acid, one preparation of BTL2 could be used to produce the S-isomer, while other preparation produced the R-isomer.

Preparation of a Stable Biocatalyst of Bovine Liver Catalase Using Immobilization and Postimmobilization Techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi‐subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross‐linking of previously immobilized enzyme with tailor‐made dextran‐aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl‐agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl‐agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross‐linked with dextran‐aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.

Purification, Immobilization, and Stabilization of a Lipase from Bacillus thermocatenulatus by Interfacial Adsorption on Hydrophobic Supports

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS‐PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl‐Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl‐Sepabeads‐BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 °C or 30% of dioxane and 45 °C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl‐Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 °C.

A Novel Heterofunctional Epoxy‐Amino Sepabeads for a New Enzyme Immobilization Protocol: Immobilization‐Stabilization of β‐Galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino‐epoxy support (amino‐epoxy‐Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the β‐galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12‐fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).